Structure of cryptophyte photosystem II–light-harvesting antennae supercomplex

Cryptophytes are ancestral photosynthetic organisms evolved from red algae through secondary endosymbiosis. They have developed alloxanthin-chlorophyll a/c2-binding proteins (ACPs) as light-harvesting complexes (LHCs). The distinctive properties of cryptophytes contribute to efficient oxygenic photosynthesis and underscore the evolutionary relationships of red-lineage plastids. Here we present the cryo-electron microscopy structure of the Photosystem II (PSII)–ACPII supercomplex from the cryptophyte Chroomonas placoidea. The structure includes a PSII dimer and twelve ACPII monomers forming four linear trimers. These trimers structurally resemble red algae LHCs and cryptophyte ACPI trimers that associate with Photosystem I (PSI), suggesting their close evolutionary links. We also determine a Chl a-binding subunit, Psb-γ, essential for stabilizing PSII–ACPII association. Furthermore, computational calculation provides insights into the excitation energy transfer pathways. Our study lays a solid structural foundation for understanding the light-energy capture and transfer in cryptophyte PSII–ACPII, evolutionary variations in PSII–LHCII, and the origin of red-lineage LHCIIs.

The analysis of the structure, especially of the Psb-\gamma protein, is interesting; the latter likely represents the most important finding of the article.A direct mapping of the PDB chain names to the chain names in the paper would have however been helpful, if only mentioning "chains N and n" in the context of Psb-\gamma, for example.
The EET analysis yields the expected results of strong coupling between the complexes.However, the corresponding method section lacks the references for the employed quantum chemistry (CAM-B3LYP and the 6-31G* basis).It is also required that some articles are cited which have used the combination of CAM-B3LYP/6-31G* for light harvesting systems before -otherwise, the choice of methods is arbitrary and would require benchmarking, which is not the article's scope.Please give the appropriate credit to previous computational work just as you would do for experimental work.
List of minor remarks: Abstract: "primitive" is an inappropriate way to classify an organism.Please use a synonym that does not suggest inferiority compared to "higher/advanced" (the opposite of "primitive") organisms.Fig. 5: "Con(n)ector" Reviewer #2: Remarks to the Author: The manuscript by Zhang et al. describes a high-resolution cryoEM structure of photosystem II from the cryptophyte Chroomonas placoidea.They found that cryptophyte PSII shows an unusual arrangement of ACPII complexes as linear trimers.They also uncovered a novel pigment binding subunit termed Psb-γ, connecting ACPII to PSII core.The manuscript discusses the evolutionary aspect of PSII across algae.Although high-resolution structures of red alga and diatom PSII were reported previously, the current work shows novel features of PSII and undisclosed binding mode of the antennae to the core complex.The data presented here is solid, however in my view the manuscript and the data analysis need to be improved before considering it for publication.

Major comments
The oxygen evolution activity needs to be presented in the manuscript or in the supplementary data to demonstrate the active complex was purified.
How were the locations of chlorophyll c2 and alloxanthins determined?Many of the map densities are not conclusive.Have you used any supporting analysis to determine their positions in the ACPIIs.
Structural flexibility -have you tried 3Dflex analysis, focused refinement, or multibody refinement?Is it possible there are additional compositional states that were not separated during classification?These would be important for ACPII-4/5/6 which have lower quality map densities.
Have you applied symmetry in the refinement process?If so, please state where it was applied in the data processing workflow and if C1 yielded a similar map.
There are many densities close to the OEC but there are no water molecules present in the model.At this resolution water molecules should be identified.Please add these to the model and compare the water network with other PSII structures from various origins.
QB was modelled in although the density does not show it is there definitively, but nonetheless it is very interesting to see it there.Can you compare it with other structures where QB is present?(Diatoms, green algae, cyanobacteria) PsbW is a single transmembrane subunit connecting the core with LHC subunits.Its location in diatom PSII is identical to PsbW in green algae (PDB 6KAF for example).Please discuss their evolutionary relationship.
Lines 152-153 -there is also a lower capacity in the orange absorption spectra (500-550nm) and a slight red-shift in the red absorption.Is this the result of chlorophyll c2 or alloxanthin and how it coincides with chlorophyll c2 blue-shift in the red absorption spectra?Please discuss the potential effect of these pigments on excitation energy transfer in PSII-ACPII.
Lines 153-157 -the Chl-binding sites are largely conserved among all ACPIIs -please add a comparison with green algae LHCII sites for both chlorophylls and carotenoids.
Possible energy transfer pathways within the PSII-ACPII supercomplex -the analysis needs to be improved by adding exact distances when comparing the pigment locations with Chaetoceros gracilis and Cyclotella meneghiniana.I did not find any reference to the EET contribution of chlorophyll c2, its energy transfer coefficient and a proposed role for its presence in the ACPII complexes.
Line 129 -"However, there are variations in the location and orientation of the β-turns (K63-E71)": in the model the residue numbers is K101-E109, please clarify which is correct.
Line 155 -"The Chl 314/315 were close to the PSII core" -please note what is the shortest distance of these to the core pigments.Line 160 -"..BC loop and the N-terminal loop.." -please state these are ACPII loops.Line 235 -"..might provide the foundation for cryptophytes and diatoms to thrive in their specific environmental niches".Please add a short explanation on how different their niches are and what would be the added value of their unique pigments.
Line 402 -which chlorophyll atoms were used to calculate the vector of the spectral overlap?
Figure 5 -please add the subunits marked in the figure and their colors to the legend.In the figure should be "Connector" and not "Conector".Supplementary table 1 -please add the map vs model CC.
1.The introduction draws an analogy between all PSII systems, by claiming them to form "PSII-LHCII supercomplexes".Phycobilisomes are usually not classified as LHCII proteins, which prevents such a generic classification when also including cyanobacteria.Reply: Thanks for pointing this out.We have revised the relevant descriptions in INTRODUCTION to distinguish between phycobilisomes and LHCII (Page 3).
2. The analysis of the structure, especially of the Psb-\gamma protein, is interesting; the latter likely represents the most important finding of the article.A direct mapping of the PDB chain names to the chain names in the paper would have however been helpful, if only mentioning "chains N and n" in the context of Psb-\gamma, for example.Reply: Thanks for the suggestion.We have added a direct mapping of the PDB chain names to the chain names in the paper in Table S3.The chain names in the paper have been labelled by the PDB chain names in the revised manuscript.
3. The EET analysis yields the expected results of strong coupling between the complexes.However, the corresponding method section lacks the references for the employed quantum chemistry (CAM-B3LYP and the 6-31G* basis).It is also required that some articles are cited which have used the combination of CAM-B3LYP/6-31G* for light harvesting systems before -otherwise, the choice of methods is arbitrary and would require benchmarking, which is not the article's scope.Please give the appropriate credit to previous computational work just as you would do for experimental work.Reply: We have added the reference (Yanai et al., Chem.Phys.Lett.2004, 393: 51-57) for the employed quantum chemistry and cited previous articles which have used this quantum chemistry method to make the description and discussion more rigorous (Page 17).
List of minor remarks: 4. Abstract: "primitive" is an inappropriate way to classify an organism.Please use a synonym that does not suggest inferiority compared to "higher/advanced" (the opposite of "primitive") organisms.Reply: We have replaced "primitive" with " ancestral ". 5. Fig. 5: "Con(n)ector" Reply: We have corrected the typo in Fig. 5.

Reviewer #2 (Remarks to the Author):
The manuscript by Zhang et al. describes a high-resolution cryoEM structure of photosystem II from the cryptophyte Chroomonas placoidea.They found that cryptophyte PSII shows an unusual arrangement of ACPII complexes as linear trimers.They also uncovered a novel pigment binding subunit termed Psb-γ, connecting ACPII to PSII core.The manuscript discusses the evolutionary aspect of PSII across algae.Although high-resolution structures of red alga and diatom PSII were reported previously, the current work shows novel features of PSII and undisclosed binding mode of the antennae to the core complex.The data presented here is solid, however in my view the manuscript and the data analysis need to be improved before considering it for publication.Reply: We sincerely appreciate Reviewer 2's highly positive comments on our work.
Major comments 1.The oxygen evolution activity needs to be presented in the manuscript or in the supplementary data to demonstrate the active complex was purified.Reply: We have measured the oxygen-evolving activity to verify the active state of the purified PSII-ACPII supercomplexes.The oxygen-evolving activity of purified PSII-ACPII was 128 ± 13 μmol O2 (mg Chl) −1 h −1 from three replicates, comparable to the activities of reported PSII complexes ( 2. How were the locations of chlorophyll c2 and alloxanthins determined?Many of the map densities are not conclusive.Have you used any supporting analysis to determine their positions in the ACPIIs.Reply: As described in METHODS (Page 15), Chl c was assigned based on the absence of density map corresponding to the phytol chain for Chl a as well as the planarity of C-18 1 , C-18, C-17, and C-17 1 resulting from the C-18=C-17 double bound for Chl c. Alloxanthin was assigned based on the density covering the two end groups shown with a threshold of 12 σ contour level as alloxanthin has more areas for the hydroxy (Fig. S3c).
3. Structural flexibility -have you tried 3Dflex analysis, focused refinement, or multibody refinement?Is it possible there are additional compositional states that were not separated during classification?These would be important for ACPII-4/5/6 which have lower quality map densities.Reply: We thank the reviewer for pointing out this important question.To address this question, we performed 3D variability analysis.The particles from different clusters were combined, and then particle subtraction was carried out, followed by local refinement with a focus on the peripheral subunits.We conducted analysis with masks of different sizes and positions for local search, particularly focusing on ACPII-6.By implementing local refinement, the resolution of the peripheral regions of the map was enhanced, resulting in an overall improvement in the quality of the structural model.The results revealed that the peripheral ACPII subunits exhibit a high degree of orientational flexibility, highlighting the dynamic architecture of PSII-ACPII.The data process and findings are illustrated in Figure R1   4. Have you applied symmetry in the refinement process?If so, please state where it was applied in the data processing workflow and if C1 yielded a similar map.Reply: We conducted Ab-Initio Reconstruction and Heterogeneous 3D Refinement with C1 symmetry, followed by Homogeneous Refinement with C2 symmetry.We have revised the data processing workflow as shown in Fig. S2.Additionally, we performed Homogeneous Refinement with C1 symmetry and compared the results in Chimera (Fig. R2 below).In the C1 symmetry map, the density appears slightly better at one end.However, in the C2 symmetry map, the averaging process resulted in consistent density across both parts of the map.Nevertheless, the two maps exhibit considerable overlap, indicating their similarity.5.There are many densities close to the OEC but there are no water molecules present in the model.At this resolution water molecules should be identified.Please add these to the model and compare the water network with other PSII structures from various origins.Reply: We tried automated addition of water molecules using software, but the map density was not sufficient to support this process.Hence, we manually incorporated water molecules into the Oxygen-Evolving Complex (OEC) area using Coot, based on the density and the comparison with reported PSII structures from cyanobacteria, red algae, and diatoms (Fig. R3, Fig. S4d).The water molecules have been added to the final structural model.We found that most cryo-EM structures could not provide sufficient density resolution for the identification of water molecules, whereas crystal structures provided better insights into water molecule distribution.Overlay comparisons reveal that the positions of OEC are conserved, and the distributions of most water molecules around OEC in the models are also similar, supporting the high conservation of the PSII core.We have added relevant descriptions in RESULTS AND DISCUSSION (Page 5) of the revised manuscript.Representative areas have been encircled to illustrate the relative positions between the two images.
6. QB was modelled in although the density does not show it is there definitively, but nonetheless it is very interesting to see it there.Can you compare it with other structures where QB is present?(Diatoms, green algae, cyanobacteria) Reply: We have compared the locations and structures of QA and QB in cryptophyte PSII with those in PSII-LHCIIs of cyanobacteria, red alga, green alga, and diatom (Fig. R4, Fig. S4c).The locations and structures of QA and the head group of QB are highly conserved.In contrast, the tails of QB possess diverse conformations and orientations, suggesting the conformational flexibility of QB compared to QA in different PSII-LHCII structures.We have added relevant descriptions in RESULTS AND DISCUSSION (Page 4-5) of the revised manuscript.7. PsbW is a single transmembrane subunit connecting the PSII core with LHC subunits.Its location in diatom PSII is identical to PsbW in green algae (PDB 6KAF for example).Please discuss their evolutionary relationship.Reply: We have compared the sequences of PsbW subunits in the PSII structures of cryptophyte, red algae, diatom, and green algae (Fig R5a, Fig. S6d).The result showed that PsbW of green algal PSII has a low sequence identity with those of red algal PSII-LHCII, cryptophyte PSII-ACPII, and diatom PSII-FCPII.Phylogenetic analysis revealed that green algal PsbW exhibits a distant evolutionary relationship with PsbW of red algae, cryptophytes, and diatoms (Fig. R5b, Fig. S6e).We have added relevant descriptions in RESULTS AND DISCUSSION (Page 6) of the revised manuscript.
8. Lines 152-153 -there is also a lower capacity in the orange absorption spectra (500-550nm) and a slight red-shift in the red absorption.Is this the result of chlorophyll c2 or alloxanthin and how it coincides with chlorophyll c2 blue-shift in the red absorption spectra?Please discuss the potential effect of these pigments on excitation energy transfer in PSII-ACPII.Chl c has also been hypothesized to play a role in energy dissipation under high-light conditions (Tsujimur et al., J Phys Chem B, 2023, 127(2):505-513).Thus, Chls c and Cars may form an energyquenching system to protect PSII-ACPII from excess irradiation.We have added relevant descriptions in RESULTS AND DISCUSSION (Page 6-7) of the revised manuscript.9. Lines 153-157 -the Chl-binding sites are largely conserved among all ACPIIs -please add a comparison with green algae LHCII sites for both chlorophylls and carotenoids.Reply: We have added a comparison with green algal LHCII sites for both chlorophylls and carotenoids (Fig. R6, also Fig. S9 in the revised manuscript).Compared to green algal LHCIIs, cryptophyte ACPIIs have a very similar number of pigment binding sites, but the positions of some of these sites vary.Specifically, green algal LHCIIs have 14 conserved Chl-binding sites, whereas cryptophyte ACPIIs share the same positions with 9 of these.Moreover, green algal LHCIIs feature 4 conserved carotenoid-binding sites, whereas cryptophyte ACPIIs share 2 of them (sites 401, 403).We have added relevant descriptions in RESULTS AND DISCUSSION (Page 7) of the revised manuscript.S1.

Reviewers' Comments:
Reviewer #1: Remarks to the Author: All my concerns have been addressed; I support the publication of the article.
Reviewer #2: Remarks to the Author: The manuscript was satisfactorily revised according to the comments, and is now suitable for publication with one minor correction.
below, and have also been added in the new Fig.S2, in the main text (Page 8), and MATERIALS AND METHODS (Page 14) in the revised manuscript.

Fig. R1 .
Fig. R1.3D variability analysis and Local refinement of the peripheral subunits of the PSII-ACPII map.The gold standard Fourier shell correlation (FSC) curves of the density maps of with the criterion of 0.143.a, Representative cryo-EM images of the 3D variability analysis of peripheral subunits of PSII-ACPII.The images below show the perspective obtained by rotating the images above by 90º.b, The workflow diagram for local refinement.
Reply: The 12 ACPIIs in cryptophyte PSII-ACPII contain a total of 133 Chl a, 14 Chl c, and 60 Car (48 alloxanthin, 8 crocoxanthin, and 4 α-carotene) molecules.In contrast, PSII-FCPII of diatom Chaetoceros gracilis contains 22 FCPII antennae, binding in total 142 Chl a, 70 Chl c, and 120 Car molecules.Interestingly, the quantities of Chl a in cryptophyte ACPIIs and diatom FCPIIs are similar, whereas the amount of Chl c in FCPIIs is five times that in ACPIIs, and the amount of Car in FCPIIs is two times that in ACPIIs.The reduced content of Chl c and Car molecules in ACPIIs results in the weak absorption of cryptophyte PSII-ACPII at 500-550 nm.The large amount of Chl c in FCPIIs may lead to the blue shift of diatom PSII-FCPII in the red absorption spectra compared to PSII-ACPII.The Chl c and Car pigments enable cryptophyte PSII-ACPII absorbs light in the blue-green region which could not be effectively absorbed by Chl a.In addition, cryptophytes possess phycobiliproteins, further enhancing their absorption of green light.These spectral features allow the survival of cryptophytes in deep water, where the blue-green light can penetrate.Moreover, Chls c could transfer energy efficiently to the coupled Chls a (Croce et al., Nat Chem Biol, 2014, 10(7):492-501), and were proposed to facilitate energy transfer from Cars to Chls a (Larkum et al., Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications, 2006, pp.261-282).